quantitative multicolor super-resolution microscopy reveals ...orca.cf.ac.uk/37363/1/lehmann...

Quantitative Multicolor Super-Resolution MicroscopyReveals Tetherin HIV-1 InteractionMartin Lehmann1, Susana Rocha2, Bastien Mangeat1,3, Fabien Blanchet3, Hiroshi Uji-i2, Johan Hofkens2,

Vincent Piguet1,3*

1 Departments of Microbiology and Molecular Medicine, Dermatology and Venereology, University Hospital and Medical School of Geneva, Geneva, Switzerland,

2 Laboratory for Photochemistry and Spectroscopy, Department of Chemistry, Katholieke Universiteit Leuven, Heverlee, Belgium, 3 Department of Dermatology and

Wound Healing, Cardiff University School of Medicine and University Hospital of Wales, Cardiff, Wales, United Kingdom

Abstract

Virus assembly and interaction with host-cell proteins occur at length scales below the diffraction limit of visible light. Novelsuper-resolution microscopy techniques achieve nanometer resolution of fluorescently labeled molecules. The cellularrestriction factor tetherin (also known as CD317, BST-2 or HM1.24) inhibits the release of human immunodeficiency virus 1(HIV-1) through direct incorporation into viral membranes and is counteracted by the HIV-1 protein Vpu. For super-resolution analysis of HIV-1 and tetherin interactions, we established fluorescence labeling of HIV-1 proteins and tetherinthat preserved HIV-1 particle formation and Vpu-dependent restriction, respectively. Multicolor super-resolution microscopyrevealed important structural features of individual HIV-1 virions, virus assembly sites and their interaction with tetherin atthe plasma membrane. Tetherin localization to micro-domains was dependent on both tetherin membrane anchors.Tetherin clusters containing on average 4 to 7 tetherin dimers were visualized at HIV-1 assembly sites. Combinedbiochemical and super-resolution analysis revealed that extended tetherin dimers incorporate both N-termini intoassembling virus particles and restrict HIV-1 release. Neither tetherin domains nor HIV-1 assembly sites showed enrichmentof the raft marker GM1. Together, our super-resolution microscopy analysis of HIV-1 interactions with tetherin provides newinsights into the mechanism of tetherin-mediated HIV-1 restriction and paves the way for future studies of virus-hostinteractions.

Citation: Lehmann M, Rocha S, Mangeat B, Blanchet F, Uji-i H, et al. (2011) Quantitative Multicolor Super-Resolution Microscopy Reveals Tetherin HIV-1Interaction. PLoS Pathog 7(12): e1002456. doi:10.1371/journal.ppat.1002456

Editor: Hans-Georg Krausslich, Universitätsklinikum Heidelberg, Germany

Received May 17, 2011; Accepted November 9, 2011; Published December 15, 2011

Copyright: � 2011 Lehmann et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permitsunrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Funding: VP was supported by the Human Science Frontier Program and Swiss National Science Foundation. SR was supported by Portuguese Foundation forScience and Technology (FCT) PhD grant SFRH/BD/27265/2006. JH was supported by the long-term structural funding program ‘‘Methusalem’’ by the Flemishgovernment, the K.U. Leuven research fund (GOA 2006/2, CREA2007), Fonds voor Wetenschappelijk Onderzoek Vlaanderen (FWO grant G.0402.09) and theHerculesstichting. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Competing Interests: The authors have declared that no competing interests exist.

* E-mail: [email protected]

Introduction

Although viruses heavily depend on the host cell machinery for

their replication, they also face numerous blockades imposed by

cellular proteins at several distinct steps in their life cycle.

Recently, tetherin, an interferon-induced transmembrane protein

has been shown to restrict the release of HIV-1 [1,2] and other

enveloped viruses [3–5]. Viruses also possess several anti-tetherin

activities encoded by proteins such as HIV-1 Vpu [2,6], SIV Nefs

and Envelope (ENV) [7–9], HIV-2 ENV[10] and Karposi’s

sarcoma-associated herpesvirus K5 [5].

Tetherin possesses two membrane anchors in an unusual

topology, namely a N-terminal transmembrane (TM) domain and

a C-terminal glycophosphatidylinositol (GPI) lipid anchor, pro-

posed to mediate lipid raft interaction [11]. The extracellular

domains of two tetherin molecules form parallel cysteine-linked

coiled-coil domains [12,13]. Perez-Caballero et al. used tetherin

mutants and artificial tetherin composed of fragments of

heterologous proteins in a tetherin-like topology to demonstrate

that tetherin inhibits HIV-1 release through direct tethering of

virions to cells [13]. Direct incorporation of tetherin into HIV-1

virions was also confirmed by biochemical analysis and electron

microscopy [13–15]. HIV-1 Vpu interacts with the tetherin

transmembrane domain [6,16] and counteracts tetherin by

degradation and removal from the cell surface [2,17–19]. Through

these combined activities, Vpu impairs incorporation of tetherin

into virions and restriction [13]. Detailed analysis of tetherin

distribution in the plasma membrane, of the role of lipid rafts in

HIV-1 tetherin interactions and of the orientation and number of

tetherin molecules involved in restriction is still lacking.

HIV-1 assembly into virions of 100–150 nm diameter at the

plasma membrane of infected cells involves an extensive range of

host cell factors [20]. Widely used electron microscopy techniques

provide detailed pictures of viral and cellular structures, but high

density labeling of viral and cellular proteins as well as quantitative

image analysis remain challenging. Novel single-molecule super-

resolution imaging by photoactivated localization microscopy

(PALM) [21], fluorescence PALM (fPALM) [22], stochastic optical

reconstruction microscopy (STORM) [23] and direct STORM

(dSTORM) [24] exploit photoswitching properties of photoacti-

vatable fluorescent proteins (PAFP) and organic dyes to localize

them with nanometer resolution. Multicolor super-resolution

microscopy [25,26] can resolve distances of 20–200 nm that are

relevant for virus-host interactions and bridge the gap between

PLoS Pathogens | www.plospathogens.org 1 December 2011 | Volume 7 | Issue 12 | e1002456

Fluorescence Resonance Energy transfer (FRET) and convention-

al diffraction limited fluorescence microscopy [27].

Previous super-resolution imaging revealed single molecule

dynamics and assembly of tandem-EosFP tagged HIV-1 Gag into

virus-like particles of 100–200 nm [21,28].

Here, we set up labeling of HIV-1 and tetherin with monomeric

PAFP and antibody staining for multicolor super-resolution

microscopy in cells. We visualized precise localization of HIV-1

proteins in virions and at budding sites at the plasma membrane of

cells. Super-resolution analysis showed that tetherin formed

clusters, whose integrity depended on both membrane anchors.

Importantly, tetherin clusters closely associated with HIV-1

budding sites. Through a combination of biochemical analysis

and super-resolution microscopy of tetherin mutants, we showed

that tetherin restricts virion release as extended dimers and that

the transmembrane domain of tetherin possesses affinity for HIV-1

assembly sites.

Results

In order to perform single-molecule super-resolution microsco-

py of HIV-1, we fused different PAFP to HIV-1 Gag, the major

structural protein of virions, yielding: Gag-Dronpa, Gag-PS-CFP2,

Gag-Dendra2, Gag-mKikGR, Gag-mEosFP and Gag-PAm-

Cherry (Figure S1A). The monomeric PAFP are expected to

minimally interfere with HIV-1 particle formation [29,30], a

potential caveat of the Gag fused to tandem-EosFP. The different

constructs were expressed together with full-length HIV-1 in 293T

cells and analyzed for incorporation into HIV-1 particles, effect on

viral infectivity and performance in single-color super-resolution

imaging [31]. We selected Gag-Dronpa and Gag-mEosFP for

super-resolution imaging of HIV-1 virions, due to their expression

as full length fusion proteins, minimal impact on infectivity and

superior signal-to-noise ratios (Figure S1B–D).

To visualize HIV-1 and cellular proteins labeled with PAFP or

Alexa Fluor 647 labeled antibodies, we setup a two-color super-

resolution microscope with widefield illumination in total internal

reflection (TIR) mode. Fluorescence emission of Dronpa, mEosFP

and Alexa Fluor 647 were detected simultaneously by two

Electron Multiplying CCD cameras using specific filter sets and

synchronized photoactivation/excitation/detection schemes as

depicted in Figure S2. Notably, differences in alignment of both

cameras and chromatic aberrations were corrected using a high

resolution mapping procedure (Figure S3) based on local weighted

mean transformation [32]. Colocalization precision of ,17 nmthroughout the total field of view was routinely achieved (Figure

S3F). Our two-color super-resolution microscope therefore

enabled colocalization analysis of proteins labeled with PAFP or

antibodies in the range of 20–200 nm which covers scales relevant

to HIV-1 host interactions.

HIV-1 virion structure and assembly sitesTo determine the localization of HIV-1 proteins within virions,

we performed super-resolution microscopy on double-labeled

HIV-1 particles.

HIV-1 virions specifically incorporated Dronpa-labeled Vpr, a

HIV-1 protein associated with viral cores, Gag-Dronpa and Gag-

mEosFP, but not Dronpa alone (Figure 1A and Figure S4A).

Notably, labeling HIV-1 virions with the monomeric PAFP fusions

minimally affected infectivity and therefore likely preserves virion

structure (Figure 1B).

HIV-1 virions that contained HA-tagged integrase (INHA) and

Dronpa-Vpr were fixed on coverslips, permeabilized and further

labeled by indirect immunofluorescence against Integrase (IN),

capsid (CA), matrix (MA) or gp120 envelope (ENV) followed by

Alexa Fluor 647-coupled secondary antibodies (Figure 1C–E).

Super-resolution microscopy of double labeled virus particles

showed an important increase in resolution when compared to

diffraction-limited confocal laser scanning or total internal

reflection fluorescence (TIRF) microscopy (Figure 1C and Figure

S1D). The high degree of colocalization in super-resolution images

of the two HIV-1 proteins Dronpa-Vpr and CA (Figure 1C)

demonstrated the performance of the calibration procedure in a

biological context. The sizes of HIV-1 structures were determined

through cluster analysis of single molecule localizations as

described in Materials and Methods after testing on simulated

clusters (Figure S5). We found similar average sizes for Dronpa-

Vpr (94617 nm), Gag-Dronpa (108639 nm) and Gag-mEosFP(94637 nm, mean6StD) consistent with their localization insideHIV-1 virions (Figure S4B).

In contrast, IN, CA, MA and ENV showed more variable

sizes: 75620 nm for IN, 112631 nm for CA, 117645 nm forMA and 127645 nm for ENV (mean6StD, Figure 1D andFigure S4C). Notably, IN colocalized with Dronpa–Vpr cores as

a discrete structure with narrow size distributions, likely due to

their common presence in the viral core. Similar sizes observed

for CA and MA structures could result from proximity of the C-

terminal epitope of mature MA and the epitope recognized by

the CA antibody. In contrast, HIV-1 ENV was found in 1-2

discrete peripheral clusters per Dronpa-Vpr containing virion

(Figure 1E).

Next, we used super-resolution microscopy to visualize HIV-1

in a cellular context. We analyzed HIV-1 protein distribution at

the plasma membrane of non-permeabilized HeLa cells transfect-

ed with HIV-1 and Gag-mEosFP and stained for ENV. We

observed distinct clusters of Gag-mEosFP surrounded by antibody

labeled ENV clusters (Figure 1F) that represent HIV-1 assembly

sites [21,28,29]. Altogether, our data demonstrates that super-

resolution microscopy allows precise localization of HIV-1

proteins in infectious virions and budding structures, which

previously could only be observed by electron microscopy [33–35].

Author Summary

Human immunodeficiency virus 1 (HIV-1) assembles andinteracts with cellular proteins at the plasma membrane ofinfected cells. Here, we analyzed individual HIV-1 virions,viral assembly sites and the mechanism of tetherinrestriction by multicolor super-resolution microscopyusing fully functional fluorescently labeled tetherin andviral proteins. Viral proteins within virions were visualizedwith nanometer resolution yielding new insight into thestructure of the HIV-1. Our super-resolution analysis wasextended to tetherin, a cellular restriction factor thatinhibits the release of several enveloped viruses. Tetherinwas localized in clusters of 70–90 nm at the plasmamembrane that contain 5–11 dimers. In contrast tetherinclusters found at HIV-1 assembly sites contained onaverage 4–7 tetherin dimers. Clustering of tetherin wasdependent on both tetherin membrane anchors. Thetransmembrane domain of tetherin associated withbudding virions independently of GM1 lipid raft domains.Our data indicated that extended dimers tether HIV-1virions directly to the cell. Overall, we provide for the firsttime super-resolution analysis of authentic virions, virusbudding sites and HIV-1 interactions with the anti-viralfactor tetherin. Our data offer novel insights into themechanisms of tetherin restriction.

Super-Resolution Microscopy of HIV-1 and Tetherin


Figure 1. Structural features of single HIV-1 virions and assembly sites are revealed by multicolor super-resolution microscopy. (A)Western blot analysis (anti-p24CA upper, anti-Dronpa bottom) of cellular lysates and purified virions from 293T cells transfected with HIV-1 INHA andindicated photoactivatable fluorescent protein (PAFP). Sizes of molecular weight markers are shown in kilodaltons. (B) Relative infectivity of virionsfrom (A). Error bars represent standard deviation of triplicate titrations. One representative experiment out of two is shown for panel A and B. (C)Virions labeled with Dronpa-Vpr and primary anti-CA and Alexa Fluor 647 secondary antibodies were analyzed by confocal laser scanning (left) orsuper-resolution microscopy (right). (D) Virions containing Dronpa-Vpr (green) were labeled by indirect immunofluorescence with primary antibodiesagainst HA (IN), HIV-1 capsid (CA), matrix (MA) or gp120 (ENV) and Alexa Fluor 647 secondary antibodies (red). Representative super-resolutionimages from two virus preparations are shown. (E) Super-resolution image (left) of ENV (red) on Dronpa-Vpr particles (green). Quantification (right)was performed by counting the number of ENV clusters on 1000 HIV-1 particles in 4 images of two independent virus preparation. (F) HeLa cells



Tetherin distribution and orientationWe next set out to visualize HIV-1 tetherin interactions at the

plasma membrane using this technique. First, the plasma

membrane distribution of endogenous and overexpressed tetherin

was analyzed by super-resolution microscopy in cells. Tetherin

constructs containing N-terminal mEosFP or epitope-tags (HA or

Flag) inserted after the extracellular coiled-coil domain (Figure 2A)

efficiently restricted the release of vpu-deficient HIV-1 (HIV-1

Dvpu) and are counteracted by Vpu in transfected 293T cells(Figure 2B and Figure S6A). Notably the cellular levels of mEosFP-

tetherin, tetherin-Flag and tetherin-HA were reduced in presence

of HIV-1 Vpu (Figure 2C and Figure S6B), indicating efficient

Vpu-mediated degradation of these constructs. The N-terminal

fusion of mEosFP to tetherin does not interfere with HIV-1

assembly (Figure S6C) and no cleavage of the fusion protein was

observed (Figure 2C and Figure S6C). Overall, labeling tetherin

with PAFP and epitope-tags minimally affects restriction activity

and preserves Vpu-sensitivity.

In HeLa cells we found endogenous and overexpressed tetherin

in homogenously distributed clusters of 70–90 nm using labeling

with mEosFP or antibody staining followed by super-resolution

microscopy (Figure 2D). Ripley’s L function was previously used to

characterize extent of clustering of influenza hemagglutinin (HA)

and T cell receptor complexes [36,37] and was tested on simulated

clusters of 50–400 nm (Figure S5). Ripley’s L function indicated

clustering of endogenous tetherin and all tetherin constructs tested

(Figure 2E). In contrast tetherin mutants lacking the transmem-

brane domain (delTM) or GPI anchor (delGPI) showed decreased

clustering (Figure 2F), which was confirmed by decreased peaks of

Ripley’s L function that shifted to larger distances compared to

wild-type tetherin (Figure 2G). Overall, labeling of tetherin for

super-resolution microscopy preserved restriction activity, Vpu-

sensitivity and revealed a clustered distribution, which depends on

both tetherin’s membrane anchors.

Distribution of tetherin molecules at HIV-1 assembly sitesSince super-resolution microscopy enabled us to resolve

structural features of viral particles and the distribution of tetherin

(Figures 1 and 2), we next determined where tetherin localizes with

respect to HIV-1 assembly sites. HeLa cells were cotransfected

with HIV-1 Dvpu and Gag-mEosFP and tetherin-HA or tetherin-Flag and analyzed by indirect immunofluorescence and super-

resolution microscopy. Gag-mEosFP-containing budding struc-

tures at the plasma membrane were mostly found in close

proximity with a single cluster of tetherin-HA or tetherin-Flag

(Figure 3A and B). Similarly, mEosFP-tetherin clusters were found

close to groups of HIV-1 ENV clusters at HIV-1 assembly sites

(Figure 3C). Close examination of 500 individual tetherin-positive

budding sites revealed that 80% contained a single cluster of

mEosFP-tetherin (Figure S6D). Finally bivariant Ripley’s L

function confirmed coclustering of different tetherin constructs

with either Gag-mEosFP or HIV-1 ENV at HIV-1 budding sites

(Figure 3D). We conclude that tetherin in single clusters closely

associates with HIV-1 budding sites.

Tetherin restriction mechanismAs both tetherin membrane anchors are essential for restricting

HIV-1 release [13], different orientations of tetherin dimers across

the cell and viral membrane are possible. Two models must be

considered: (i) the ‘‘extended model’’ in which pairs of membrane

anchors are incorporated into the cell membrane and the viral

membrane, and an extended coiled-coil domain spans the gap

between both membranes or (ii) the ‘‘parallel model’’ where one

tetherin monomer is incorporated into the cell membrane and the

other monomer into the viral membrane (Figure 4A). The

apparent distances of circa 17 nm found between tethered virions

by electron microscopy [6] and the structure of the tetherin

ectodomain [12] favor the extended model but definitive proof is

missing.

Tetherin-restricted virions are efficiently released from the cell

surface by treatment with subtilisin A, a protease with relatively

low specificity [1,6,14,15]. If virions are retained following the

parallel model, subtilisin A treatment should leave monomeric low

molecular weight N-termini inside stripped particles.

293T cells expressing HIV-1 Dvpu and N-terminal HA-taggedtetherin were treated with subtilisin A, cell lysates and released

virions were then analyzed by Western blot. Analysis of cellular

extracts showed constant pr55Gag content, whereas the majority

of glycosylated tetherin at 36 and 60 kDa, as well as virions

containing p24CA were efficiently removed by subtilisin A

treatment (Figure 4B). Only a HA fragment of ,26 kDa wasfound inside stripped virions by non-reducing SDS-PAGE/

Western blot analysis (Figure 4B). Under reducing SDS-PAGE a

single band migrated at ,13 kDa, consistent with a previouslyproposed cleavage site at RNVT/H68 [13,14]. Altogether, we

found dimeric N-termini associated with subtilisin A stripped

virions, which is not compatible with the parallel model of tetherin

orientation.

If tetherin retains viruses via the extended model, cleavage of

the tetherin GPI anchor by phosphatidyl-inositol-specific phos-

pholipase C (PI-PLC) should release tethered virions. To test this

hypothesis, 293T cells transfected with HIV-1 Dvpu and tetherin-HA, HA-tetherin or the inactive mutant tetherin-HA delTM

(Figure S6A and [13]) were treated with PI-PLC or subtilisin A.

Stripped virions were pelleted through sucrose and analyzed by

Western blot. Both PI-PLC and subtilisin A treatment released

virions retained by tetherin (Figure 4C). Quantification from

several experiments revealed that PI-PLC treatment (1 U/ML)

released 20% of virions compared to the maximal release by 5 mg/ML subtilisin A (Figure 4D). To compare PIPLC and subtilisin A

activities on tetherin we used wt tetherin HA and the mutant

delTM, that is attached to the cell only via a GPI anchor.

Fluorescence-activated cell sorting (FACS) analysis revealed that

PI-PLC specifically reduced cell-surface levels of tetherin-HA

delTM, whereas increased signal from tetherin-HA could result

from increased access of antibodies to the internal HA tag after

tetherin GPI cleavage (Figure 4E). Subtilisin A treatment removed

the majority of tetherins from the cell surface, indicating that lower

release of tetherin restricted virions by PI-PLC can be explained

by its lower activity in cleaving the tetherin GPI anchor when

compared to proteolysis cleavage by subtilisin A.

Finally, virions released by PI-PLC treatment contained nearly

full length dimeric tetherin (Figure 4F). Therefore, since both PI-

PLC and subtilisin A treatments removed tethered virions, which

respectively contained dimeric tetherin and dimeric N-terminal

tetherin fragments we conclude that tetherin restricts HIV-1

release as an extended dimer.

To characterize further the orientation of tetherin upon

incorporation into the membrane of assembling virions, we

expressing HIV-1 Dvpu and Gag-mEosFP (green) were labeled by indirect immunofluorescence with primary anti-gp120 (ENV) and Alexa Fluor 647secondary antibodies (red), conventional resolution (left) and super-resolution image (right), scale bars 1 mm (c) and 200 nm (d–f).doi:10.1371/journal.ppat.1002456.g001



Figure 2. Tetherin localization to micro-domains depends on both membrane anchors. (A) Schematic presentation of tetherin constructsused and labeling/photoswitching scheme of fluorescent Alexa Fluor 647 (violet) and dark state (grey), tetherin structural features are:transmembrane domain (TM), coiled-coil domain (CC) and Glycosylphosphatidylinositol (GPI) anchor. HA or Flag are internal HA or Flag tags. (B) 293Tcells were transfected with HIV-1 Dvpu and either mEosFP, mEosFP-tetherin or tetherin-Flag without or with Vpu as indicated and infectious outputwas determined on HeLa indicator cells. Error bars represent range of duplicate titrations. (C) Western blot analysis of cell lysates from B) wasperformed for pr55Gag, tetherin, Vpu, GFP as transfection control and PCNA as loading control. Sizes of molecular weight markers are shown inkilodaltons. One representative experiment out of two is shown for panel B and C. (D) Representative regions of super-resolution images of HeLa cellsexpressing HIV-1 Dvpu and empty plasmid, mEosFP-tetherin or tetherin-Flag that were labeled by indirect immunofluorescence against tetherin or



analyzed the distribution of tetherin mutants lacking one of the

membrane anchors relative to HIV-1 budding sites by super-

resolution microscopy and co cluster analysis using bivariant

Ripley’s L function. HeLa cells were transfected with HIV-1 Dvpu,Gag-mEosFP and tetherin-HA or its mutants delTM and delGPI

and labeled by anti-HA immunofluorescence. Tetherin-HA and

the delGPI mutant, but not delTM associated with Gag-mEosFP-

containing budding sites (Figure 5), indicating that the tetherin

transmembrane domain drives tetherin localization to HIV-1

budding sites.

Finally to test whether tetherin could associate with budding

structures via lipid rafts, HeLa cells expressing HIV-1 Dvpu andGag-mEosFP or mEosFP-tetherin, were fixed, stained for the lipid

raft marker GM1 using Alexa Fluor 647 Cholera-toxin and

analyzed by super-resolution microscopy and co cluster analysis

using bivariant Ripley’s L function. GM1 localized to clusters of

variable sizes that showed minor coclustering with mEosFP-

tetherin domains, but did not show significant overlap (Figure 6B

and C), indicating that both proteins are found in distinct, but

adjacent domains. In contrast, we could not detect significant

coclustering of GM1 with Gag-mEosFP-containing HIV-1 bud-

ding sites (Figure 6A and C). As positive control for coclustering,

we monitored mEosFP-tetherin and tetherin-HA at HIV-1

budding sites labeled by HIV-1 ENV and Gag-mEosFP,

respectively (Figure 3D and 6C). In addition, the GPI anchor

was not sufficient to mediate enrichment of tetherin-HA delTM at

budding sites (Figure 5). We conclude that tetherin and GM1,

although both were reported to associate with lipid rafts, localize

to different but adjacent lipid domains and that GM1-containing

lipid rafts and the tetherin GPI anchor are unlikely to drive

tetherin to HIV-1 assembly sites.

Quantification of tetherin molecules at HIV-1 assemblysites

Single-molecule imaging and localization of PAFP provides

super-resolution images and was previously used to estimate

Flag or left unlabeled (mEosFP-tetherin), scale bar 500 nm. E) Ripley’s L analysis: normalized L(r)-r plots indicate clustering at distances r with positiveL(r)-r values, F) Representative regions of super-resolution images of HeLa cells expressing HIV-1 Dvpu and indicated tetherin-HA or mutantconstructs. The cells were labeled by indirect immunofluorescence with primary anti-HA and Alexa Fluor 647 secondary antibodies, scale bar 500 nm.G) Ripley’s L analysis indicates higher degree of clustering of wt tetherin than tetherin mutants delTM and delGPI.doi:10.1371/journal.ppat.1002456.g002

Figure 3. Single tetherin domains cocluster with HIV-1 budding sites. (A–C) Representative regions of super-resolution images of HeLa cellstransfected with HIV-1 Dvpu and: (A) Gag-mEosFP and tetherin-HA; (B) Gag-mEosFP and tetherin-Flag and (C) mEosFP-tetherin. Tetherin-HA, tetherin-Flag and HIV-1 ENV were stained by indirect immunofluorescence for HA, Flag and ENV, respectively. (D) Bivariant Ripley’s L analysis indicatescoclustering of tetherin-HA, tetherin-Flag and mEosFP-tetherin with HIV-1 Gag-mEosFP and ENV, respectively. Images are representative of 5-10 cellsfrom two independent transfections.doi:10.1371/journal.ppat.1002456.g003



molecule numbers [37–39]. Detailed photophysical characteriza-

tion of the irreversible photo-convertible mEos [40] revealed long-

lived dark states of the photoactivated red form that could lead to

clustering artifacts and significant overcounting [39,41]. Annibale

et al. showed that continuous instead of pulsed photoactivation by405 nm light and the introduction of dark times significantly

reduced overcounting [41]. Following this methodology, fields of

monodispersed mEosFP were used to determine appropriate

imaging and analysis parameter including 561 nm excitation

intensity [42], continuous 405 nm photoactivation and a dark time

of 5 s. As a result, we found 1–2 reactivation events per single

mEosFP molecule (25th–75th percentile range, Figure 7C and

Figure S7), which may reflect the presence of a minor fraction of

mEosFP multimers. Subsequently, using this optimized imaging

and analysis parameters we estimated the number of mEosFP-

tetherin molecules in free clusters and at HIV-1 budding sites

(Figure 7A,B). At 550 assembly sites, we found 7–14 mEosFP-

tetherin molecules (25th–75th percentile range, median is 10),

corresponding to 4–7 tetherin dimers per budding site (Figure 7D).

In contrast 400 mEosFP-tetherin clusters in the absence of HIV-1

contained 11-22 mEosFP-tetherin molecules (25th–75th Percentile

range, median is 16), corresponding to 5–11 tetherin dimers

(Figure 7D). The difference is significant (p,0.001, Student’s t-test) and indicates that tetherin clusters can associate with HIV-1

budding sites. Approximately 70% of tetherin molecules within a

cluster stay associated with budding virions and may participate in

restricting HIV-1 release.

Discussion

We have performed biochemical and super-resolution analysis

to provide further understanding of the interactions between the

restriction factor tetherin and HIV-1.

First, to achieve specific and high density labeling of HIV-1 for

super-resolution microscopy, we tested different monomeric PAFP

in HIV-1 Gag fusions, similar to Gag-GFP that showed budding at

the plasma membrane [29,30]. Gag-Dronpa, Gag-mEosFP and

Dronpa-Vpr were incorporated into full HIV-1 particles, mini-

mally affected infectivity and enabled super-resolution microscopy

analysis of virions. This confirmed the expected virion diameter of

100 nm. Of note is the fact that Gag, fused to 50 kDa dimeric Eos

protein, produced larger virus-like particles of 100–200 nm in

initial super-resolution studies [21,28]. For multicolor super-

resolution, we labeled HIV-1/cellular proteins with Dronpa,

mEosFP or antibodies coupled to the photoswitchable dye Alexa

Fluor 647 [24,43]. Using simultaneous detection of two fluorescent

markers and high-resolution registration mapping [32] we

acquired two-color super-resolution images within few minutes

and with a colocalization precision of 17 nm throughout a field of

view. The precise localization of antibody-labeled viral proteins

within Dronpa-Vpr virions was determined by super-resolution

microscopy. In accordance with PALM and electron microscopy

[33] structures labeled with CA or MA antibodies had sizes of

112 nm and 117 nm, respectively and colocalized with Dronpa-

Vpr. In contrast, HIV-1 integrase localized to a structure with a

characteristic size of 75 nm within Dronpa-Vpr containing cores.

HIV-1 ENV was predominantly found in 1–2 peripheral clusters

close to Dronpa-Vpr, consistent with their localization in the viral

membrane. A clustered distribution of ENV on HIV-1 virions was

observed in electron tomography images and could be functionally

relevant during cell attachment and fusion [35,44].

HIV-1 assembly at the plasma membrane of HeLa cells was

previously visualized using diffraction-limited microscopy

[29,30,45]. Super-resolution microscopy of Gag-mEosFP and

antibody-stained ENV on non-permeabilized cells revealed

important structural features of budding sites. Gag-mEosFP

clusters of varying sizes together with clustered ENV were found

in the same TIRF imaging plane close to the coverslip, indicating

assembly at the plasma membrane. As previously noted, ENV was

found in distinct clusters [46] that incorporated into Gag-mEosFP

assembly sites. Overall, our super-resolution microscopy analysis

provides a detailed picture of HIV-1 virions and their assembly

sites, which was previously exclusively obtained by electron

microscopy.

We applied super-resolution microscopy for the HIV-1 cellular

restriction factor, tetherin. Endogenous and overexpressed tetherin

were previously identified in endosomal compartments and in

clusters at the plasma membrane both by fluorescence microscopy

[2,3,12,15] and electron microscopy [14,15,47]. The overall

clustered distribution of tetherin was not influenced by HIV-1

particle formation, however localization of tetherin at budding

sites was noted in some cases [2,3,14,15], but not all [18,48]. The

size of the clusters could not be determined due to the diffraction-

limited resolution of conventional fluorescence microscopy or low

density labeling in immuno-electron microscopy.

Using different labeling approaches for super-resolution mi-

croscopy and calibrated size measurements we found that both

endogenous and overexpressed tetherins are organized in 70–

90 nm clusters at the plasma membrane of HeLa cells. Annibale

et al recently reported that dark state recovery of mEos2 can cause

clustering artifacts, due to repeated localization of single molecules

[41]. Since we observe similar cluster sizes for all tetherin

constructs and endogenous tetherin, consistent clustering of

mEosFP-tetherin using comparable imaging parameter as [41]

and a significant decrease in clustering of different mutant

Figure 4. Tetherin restricts HIV-1 release as extended dimers. (A) Model of tetherin, enzymatic cleavage sites of subtilisin A (SubtA) andphosphatidyl-inositol-specific phospholipase C (PI-PLC) and possible orientations during restriction, disulfide bonds are represented in red. (B)Western blot analysis of subtilisin A stripped cells and virions. 293T cells transfected with HIV-1 Dvpu and HA-tetherin were treated with subtilisin A(0,5 or 50 mg/ML). Released Virions were pelleted through sucrose and analyzed under non-reducing (-bME) or reducing (+bME) SDS-PAGE/Westernblot together with corresponding cell lysates (+bME). Data is representative of 2 experiments. (C) Western blot analysis (anti-p24CA) of purified virionsreleased from non-treated (nt), PI-PLC (1 U/ML) or subtilisin A (0.2, 1 or 5 mg/ML) treated 293T cells transfected with HIV-1 Dvpu, GFP and eithertransmembrane-deficient tetherin-HA delTM, tetherin-HA or HA-tetherin, that contain either a C or N-termal HA-tag. Numbers below each laneindicate integrated densities in arbitrary units and are representative of 3 experiments. (D) Quantification of viral release from non-treated (nt), PI-PLCor subtilisin A treated cells 3-4 independent experiments as in B), maximal release by 5 mg/ML Subtilin A treatment was normalized to 100%. Errorbars represent standard deviations and * indicates statistically significant difference with p = 0.05 (two-tailed paired Student’s t-test). (E) Enzymaticremoval of tetherin from cells in (C) was analyzed by Fluorescence-activated cell sorting (FACS). Shown are mean fluorescence intensities of anti-HAlabeling in GFP-positive cells with non-treated cells set to 100%. Error bars represent standard deviations (n = 3). (F) Western blot analysis of PI-PLCstripped virions from 293T cells transfected with HIV-1 Dvpu and HA-tetherin or tetherin-HA. Virions that were constitutively released (const.) orreleased following incubation of cells with 1 U/ML PI-PLC were pelleted through sucrose. Virions and corresponding cell lysates were analyzed byWestern blotting with anti-HA, anti-p24CA and anti-actin antibodies. Numbers below each lane indicate integrated densities of p24CA in arbitraryunits. Sizes of molecular weight markers are shown in kilodaltons.doi:10.1371/journal.ppat.1002456.g004



tetherins we report here a relevant localization of tetherin at the

plasma membrane.

Notably, our increased resolution revealed that both membrane

anchors of tetherin are required for clustering possibly through

high order complexes between dimers [13], interaction of the

cytoplasmic tail with the actin cytoskeleton [49] and lipid raft

association via the GPI anchor [11].

Several models depicting the orientation of tetherin during the

restriction on release of enveloped viruses have been proposed [6].

Here, we provide biochemical and microscopic evidence for HIV-

1 restriction via an extended conformation of tetherin dimers. This

model requires that pairs of membrane anchors incorporate into

the cell membrane and the viral membrane and an extended

coiled-coil domain spans the gap between both membranes

(Figure 4). PI-PLC and subtilisin A treatment of 293T cells

transfected with tetherin efficiently removed virions from the

surface that contained dimeric tetherins. Of note, vpu-deficient

HIV-1 virions were not released by PI-PLC treatment from HeLa

cells, but efficient cleavage of the tetherin GPI anchor was not

demonstrated [15,50]. Furthermore, slower enzymatic cleavage of

the GPI anchor could occur in virion associated clusters of tetherin

compared to efficient proteolytic cleavage by subtilisin A. In HeLa

cells higher endocytosis rates could result in lower amounts of vpu-

deficient virions bound to the cell surface compared to 293T cells

[1]. Virus accumulation in biofilm-like extracellular assemblies

[51] could further limit stripping efficiency by PI-PLC. Alterna-

tively a fraction of tetherin that contains a second transmembrane

domain instead a C-terminal GPI-anchor would be insensitive to

PI-PLC treatment [50]. Since our results and previous reports

indicate a C-terminal GPI-modification of rat and human tetherin

[11,13,52] further biochemical analysis of tetherin C-terminal

membrane anchor by mass spectroscopy is needed.

Figure 5. Association of tetherin with HIV-1 budding sites depends on its transmembrane domain. A) Representative regions of super-resolution images of HeLa cells transfected with HIV-1Dvpu, Gag-mEosFP and tetherin-HA wt, delTM or delGPI, scale bar 200 nm, B) bivariant Ripley’sL analysis on fields shown in A), C) Multiple fields were analyzed by bivariant Ripley’s L function and r(max) and L(max) determined, error barsrepresent standard deviations (9 fields from 3 cells per conditions), p values were determined by Student’s t-test.doi:10.1371/journal.ppat.1002456.g005



Of note, our biochemical analysis does not provide information

on initial orientation of extended tetherin dimer at assembly sites

since chains and clusters of tethered HIV-1 particles contain both

of the possible orientations (Figure 4A) [13].

Therefore, we compared the incorporation of tetherin mutants

lacking either the TM or GPI membrane anchor into single

budding sites in HeLa cells by super-resolution microscopy. We

found that the tetherin transmembrane domain stably associated

with HIV-1 membranes during assembly.

Alternatively tetherin could also associate with HIV-1 budding

sites via shared localization to lipid raft domains as both show

some resistance to cold detergent extraction [1,11,53,54]. Both

tetherin and Gag also cofractionated with the lipid raft marker

caveolin [55]. Nevertheless cofractionation of proteins with raft

markers does not prove their direct association or localization to

similar lipid raft domains [56]. Indeed crosslinking antibodies

against tetherin inhibited its antiviral effect but increased tetherin/

Gag cofractionation [55].

Using super-resolution microscopy we found tetherin clusters in

close association with GM1 lipid domains, but without significant

overlap as previously noted for other raft proteins [57]. HIV-1

budding sites did not show significant association with GM1 lipid

domains or with a tetherin mutant containing only the GPI

membrane anchor. Recently HIV-1 Gag multimerization was

shown to induce the coalescence of lipid raft markers and

tetraspanins as visualized by antibody copatching, FRET analysis

and single molecule tracking [58,59]. Both studies required

copatching of raft markers (GM1, CD55 and HA–TM) and

tetraspanins (CD9, CD81) to demonstrate their enrichment at

HIV-1 budding sites due to the limited detection sensitivity of

conventional fluorescence microscopy. Copatching could affect

protein mobility and association with HIV-1 budding sites. GFP-

GPI and unpatched CD55 failed to stably associate with viral

assembly sites [45,59]. The later observations are consistent with

our results obtained from unpatched GM-1 that was stained after

fixation. Additionally the stable association of patched HA-TM

and tetraspanins with viral assembly sites [58,59] is in line with our

observation that clustered tetherin molecules stably associates with

the viral membrane via their transmembrane domains.

Altogether the tetherin GPI modification and the association

with GM1 lipid domains are not important for localization of

tetherin to HIV-1 budding sites, but implicated in local

concentration of tetherin (Figure 2) and maybe important for the

endocytosis of retained virions. Alternatively tetherin could

associate with HIV-1 assembly sites via other lipid rafts or

tetraspanin-enriched domains. More efficient tetherin restriction

could be obtained via late and stable incorporation into budding

membranes within tetraspanin-enriched domains compared to

early or transient association observed with raft markers [58,59].

At HIV-1 assembly sites, we found single tetherin clusters.

mEosFP-tetherin showed a clustered plasma membrane distribu-

tion, restricted efficiently HIV-1 particle release in a Vpu-

dependent manner (Figure 2A,B and Figure S6) and did not

interfere with Gag assembly (Figure S6A). Therefore, the

irreversible photoswitching properties of mEosFP allowed us to

determine relevant mEosFP-tetherin quantities at HIV-1 assembly

sites. Careful photo-physical characterization of purified mEosFP

and the adjustment of imaging and analysis parameters enabled

reliable single molecule counting of mEosFP.

We found 5–11 tetherin dimers in single clusters in the absence

of HIV-1 and 4–7 tetherin dimers associated with HIV-1 budding

sites, that represents a significant difference (p,0.001, Student’s t-test). This indicates that about 70% of tetherin molecules within a

cluster remain stably associated with budding sites possibly

through incorporation of their TM domains into the viral

membrane. Overall a low number of clustered tetherin dimers is

sufficient to restrict the release of newly formed virions.

Interestingly crosslinking antibodies against tetherin interfere with

tethering function, reduce incorporation of tetherin into virions

and affected the distribution of tetherin within membrane raft

fractions [55]. Therefore it is possible that antibody crosslinking

affects tetherin clustering, but cannot be detected by conventional

diffraction limited microscopy [55]. We propose that the

supramolecular organization of tetherin dimers in clusters could

concentrate and position tetherin for optimal restriction and limit

access to viral countermeasures. In summary, our biochemical and

super-resolution analysis provided new insights into tetherin

interaction with HIV-1 virions. We can propose the following

mechanism for HIV-1 restriction by tetherin: Initially, tetherin

locally concentrates in clusters containing 5–11 dimers and this

involves both TM and GPI membrane anchors. In the absence of

Vpu, tetherin N-termini associate with HIV-1 budding sites

independently of GM1-enriched raft domains and become

trapped during Gag-multimerization. Clusters containing 4 to 7

tetherin dimers remain associated with budding virions and can

mediate restriction. Flexible coiled-coil interactions within dimers

[12] are likely to enable retention of GPI anchors within the host

cell membrane during budding and membrane scission. The final

Figure 6. Tetherin domains and HIV-1 budding sites do not overlap with GM1 containing lipid rafts. (A,B) Representative regions ofsuper-resolution images of HeLa cells transfected with HIV-1 Dvpu and Gag-mEosFP(A) or mEosFP-tetherin (B) that were fixed and stained for GM1with Cholera toxin B Alexa Fluor 647, scale bars 200 nm (C) Bivariant Ripley’s L analysis of panel A and B of this figure and Figure 3A and C Images andcocluster analysis shown is representative of multiple fields from 4–6 transfected cells from two independent transfections.doi:10.1371/journal.ppat.1002456.g006



Figure 7. Single fluorescent molecule quantification of mEosFP-tetherin at HIV-1 budding sites. (A) Representative region of super-resolution images of HeLa cells transfected with HIV-1 Dvpu and mEosFP-tetherin and stained by indirect immunofluorescence for HIV-1 ENV.Acquisition was performed under alternating 561 and 644 nm excitation and continuous 405 nm photoactivation for 15 000 frames, scale bar200 nm. (B) Intensity traces of single mEosFP-tetherin clusters as depicted in (A). Automated detection of activation events on top of each trace isdepicted using a threshold of 10 standard deviations above background and a dark time of 5 s. (C) Histogram of number of activation events persingle mEosFP molecules in 1% PVA (n = 125). (D) Histogram of number of activation events of mEosFP-tetherin molecules in free clusters (n = 400) or



predicted topology is that of extended tetherin dimers with N-

termini inside newly formed virions.

Our study demonstrates that multicolor super-resolution

imaging allows characterization of the interplay between viral

and cellular structures with nanometer resolution. Quantitative

analysis of single molecule localization in combination with

biochemical analysis offers novel insights into the tetherin

restriction mechanism and can be used to investigate virus-host

interactions.

Materials and Methods

Cell linesHuman cell lines 293T, HeLa and HeLa-derived TZMbl were

obtained through NIH AIDS Research and Reference Reagent

Program and grown under standard conditions.

Plasmids and reagentsThe HIV-1 expression vectors pR9 INHA (kind gift of F.D.

Bushman, University of Pennsylvania) and vpu-deficient pR9

Dvpu (HIV-1 Dvpu) are based on pR9 [60]. Plasmids coding forGag-PAFP were obtained by replacing GFP from pGag-GFP [61]

with Dronpa [62], mKikGR [63], mutant mKikGR15.1 contain-

ing K141E and V160I (kind gifts from A. Miyawaki, Riken Brain

Science Institute, Japan), PS-CFP2, Dendra2 [64] (both from

Evrogen), mEosFP [40] (pQE32 mEosFP was a kind gift of

J.Wiedenmann, University of Southampton, U.K) or PAm-

Cherry1 [65] (kind gift of V. Verkhusha, Albert Einstein College

of Medicine, NY) by standard PCR cloning. The plasmid coding

for Dronpa, (pCDNA3 Dronpa) and Dronpa-Vpr was generated

by standard PCR cloning. ptetherin-Flag was constructed by

inserting the Flag tag after the predicted coiled-coil domain

(residue 154) by PCR cloning as previously described [3].

pmEosFP-tetherin contains mEosFP separated by a spacer peptide

(ggglyksglrsra) from tetherin N-terminus. Plasmid coding for HA-tetherin was previously described in [19]. Details for primers and

cloning can be provided upon request. Plasmids coding for

tetherin-HA as well as mutants delTM and delGPI [13] were a

kind gift from P. Bieniasz (Aaron Diamond AIDS Research

Center, NY).

Transfections, virus production and infectivity assay293T cells were transfected using a standard calcium-phos-

phate-based technique. HeLa cells were transfected using

Lipofectamine 2000 (Invitrogen) or Fugene HD (Roche), accord-

ing to manufacturer instructions. Infectious HIV-1 particles were

produced from 293T cells, filtered through a 0.45 mm filter andconcentrated through 20% sucrose by centrifugation [6]. Viral

titer was determined by applying limiting dilutions of filtered

supernatants from producer cells on HeLa TZMbl indicator cells

for 48h. Cells were fixed in 1% formaldehyde and stained for b-lactamase expression with 5-bromo-4-chloro-3-indolyl-b-D-galac-

toside (X-gal).

Protein and immunofluorescence analysisEnzymatic removal of surface virions was performed by

incubating transfected 293T cells 36 h post transfection with

1 U/ML PI-PLC (Invitrogen) in DMEM or 0.2–50 mg/MLsubtilisin A (Sigma-Aldrich) in stripping buffer [13] for 1 h at

37uC followed by filtration and concentration through 20%

sucrose. Viral pellets and cellular lysates were lysed in RIPA with

protease inhibitor (Sigma) and subjected to standard SDS Page/

Western blot analysis. Proteins were detected by mouse monoclo-

nal antibodies against Dronpa (Amalgaam), HIV-1 p24CA (183-

H12-5C), mature HIV-1 p17MA (4C9, kind gift from M. Marsh),

HA tag (Roche), GFP (Miltenyi biotec), PCNA (Oncogene

Research Products) or rabbit polyclonal antibodies against tetherin

and Vpu made by K. Strebel (obtained through NIH AIDS

Research and Reference Reagent Program).

Recombinant mEosFP was purified from E. coli BL21 DE3transformed with pQE32 mEosFP after 5 h culture at 37uC inpresence of 100 mM IPTG as described in [40]. Purified proteinwas desalted using PD10 columns (GE Healthcare) and protein

content was determined using BCA (Thermo Scientific) against

known concentrations of bovine serum albumin. For single-

molecule characterization 1 nM mEosFP was prepared in 1%

PVA and spin-coated on clean coverslips for 2 min at 3000 rpm.

For immunofluorescence analysis, HeLa cells or viral particles

on coverslips were fixed with 3% paraformaldehyde and incubated

under standard conditions with mouse monoclonal antibodies

against HA-tag (Covance), Flag-tag (Sigma), HIV-1 p24CA,

mature HIV-1 p17MA, mouse polyclonal antibody against

tetherin (kindly provided by Chugai Pharmaceutical Co., Ltd,

Kanagawa, Japan) or human monoclonal antibody against HIV-1

gp120 (2G12, obtained through NIH AIDS Research and

Reference Reagent Program). Alexa Fluor 647-conjugated sec-

ondary antibodies and Alexa Fluor 647-conjugated Cholera toxin

subunit B were purchased from Invitrogen. Super-resolution

microscopy was performed on cells and viruses in chambered

coverglass (LabTek) with freshly prepared switching buffer [24].

Super-resolution microscopySuper-resolution microscopy was performed on an inverted

microscope (Olympus IX-71) equipped with an TIRF oil objective

(60x, NA 1.6, Olympus) and two simultaneously acquiring cooled

Electron Multiplying-CCD (ImagEM, Hamamatsu). Dronpa was

excited with 1–5 kW/cm2 of 491 nm laser (Cobolt), mEosFP with

0.4 kW/cm2 of 561 nm (Cobolt) and Alexa Fluor 647 with

0.3 kW/cm2 of 644 nm (Spectra Physics) diode pumped solid state

lasers. Photoactivation of photoactivatable proteins was performed

with a 405 nm laser (Cube, coherent) using stroboscopic

illumination [31] or continuous illumination with 0.2–1.8 W/

cm2 obtained with an optical acoustic modulator (AA Opto-

Electronics). Alexa Fluor 647 was activated by 491 nm (Cobolt) or

532 nm (JP Uniphase) laser diode pumped solid state laser.

For the simultaneous measurement of Dronpa and Alexa Fluor

647, the laser lines (405, 491 and 64 nm) were combined using a

405 (zt405rdc, Chroma) and a 505 dichroic (505DCLP, Chroma)

and further guided onto the sample through the same dichroic

mirror (z488/633rdc, Chroma). When measuring mEosFP and

Alexa Fluor 647 simultaneously, the laser lines (405, 532, 561 and

644 nm) were combined using a 405 (zt405rdc, Chroma), a 532

(z532rdc, Chroma) and a 561 dichroic (z561rdc, Chroma). The

four laser lines were then guided onto the sample through the

same dichroic mirror (z561/644rdc, Chroma).

Emission from TIRF illuminated sample was collected by the

same objective and split by 650 long pass dichroic mirror (z650rdc,

Chroma). Additional filters were used to remove excitation light

and signal from other channels, namely 527/30 band pass for

Dronpa (HQ527/30m, Chroma), 570 long pass and 607/67 band

clusters that are associated with HIV-1 budding sites (n = 550) in 5 cells. Background was selected in regions without clusters. Range indicates 25th–75th percentile. *** indicates statistically significant difference with p,0.001 (two-tailed paired Student’s t-test).doi:10.1371/journal.ppat.1002456.g007



pass for mEosFP (HQ570LP and HQ607/67, Chroma), 665 long

pass and 700/71 band pass for Alexa Fluor 647 (HQ665LP and

HQ700/71x, Chroma). The images were acquired with a final

maximum field of view of ca. 41641 mm2 (80680 nm2 per pixel)with a frame rate of 10–20 Hz. In order to reduce the background

and the crosstalk between the two channels, the excitation of the

two fluorophores and data acquisition were synchronized using 2

mechanical shutters (NewPort). No additional stabilization of the

system was required. Analysis of image sequences was performed

with a homemade MATLAB routine for single molecule

localization [31].

Registration mapping for two-color imaging was performed by

a homemade Matlab routine. Briefly, fields of 100–400 fluorescent

beads (100 nm, Tetraspek, Invitrogen) were recorded as fiducial

markers in both channels. Individual bead positions were

determined by Gaussian fitting for both channels, assigned

automatically and used to calculate a local weighted mean

mapping as described in [32] and detailed in Figure S3.

Cluster and cocluster analysisFor the determination of the cluster sizes, individual clusters

were identified as discrete accumulations of 100 or more single

molecule localizations within a fixed radius of 100–200 nm. The

sizes were determined as 4 sigma of Gaussian function fitted to the

distribution of localizations. Ripley’s L function and bivariant

Ripley’s L function were determined from 464 mm2 field of viewas described [66].

Counting of mEosFP moleculesFor counting the number of mEosFP molecules per cluster

images were acquired using continuous illumination with 0.4 kW/

cm2 of 561 nm to excite the red form of mEosFP and 0.5–1.8 W/

cm2 405 nm laser light for photoactivation. Centers of individual

clusters of mEosFP-tetherin within 100 nm from ENV staining

were manually selected in two-color super-resolution images. The

intensity of 9 pixels around the cluster position was measured and

plotted against time for 159000 frames. Single molecules wereidentified in intensity traces when intensity signals increased 10

standard deviations above background [38] using a homemade

Matlab routine. Intensity increases within a dark time of 5 s were

not considered to exclude blinking and recovery from long-lived

dark states of activated mEosFP molecules.

Of note, the number of mEosFP-tetherin molecules represents a

lower estimate since photoactivation prior to visualization,

incomplete photoactivation during the experiment and missed

detection of activated molecules may not be completely excluded.

Accession numbersThe human tetherin clones used in this study correspond to

Swiss-Prot entries Q10589.

Supporting Information

Figure S1 Screening photoactivatable fluorescent pro-teins (PAFP) for HIV-1 Gag labeling and PALM. (A)Constructs used for PALM: HIV-1 Gag consisting of matrix (MA),

capsid (CA), nucleocapsid (NC) and p6 was fused to different

photoactivatable proteins, namely Dronpa, PS-CFP2, Dendra2,

mKikGR, mEosFP and PAmCherry. Color code refers to

standard emission color and gray indicates initial or photoinduced

dark state. Numbers indicate wavelengths in nm used for

photoactivation or excitation. (B) Western blot analysis (anti-

p24CA) of cellular lysates and purified virions from 293T cells

transfected with expression plasmids for HIV-1 together with

indicated HIV-1 Gag-PAFP. (C) Infectivity of released virus as

determined by single cycle replication assay in TZMBL cells and

X-gal staining, ‘‘no PAFP’’ represents 100%. Error bars represent

standard deviations (n$4), (D) Labeled virions analyzed by totalinternal reflection fluorescence microscopy (TIRF, top row) and

PALM (bottom row) for comparison purposes, scale bar 200 nm.

(TIF)

Figure S2 Setup and excitation/detection scheme usedfor super-resolution microscopy. Setup, excitation anddetection scheme used to measure Dronpa and Alexa Fluor 647

(A) or mEosFP and Alexa Fluor 647 fluorescence (B). Specific

dicroic mirrors (DM1-4) and filters (BP1-2) are described in

Material and Methods. OAM: acoustic optic modulator, MS1:

mechanical shutter synchronized with CCD1, MS2: mechanical

shutter synchronized with CCD2.

(TIF)

Figure S3 Colocalization procedure for two-color super-resolution microscopy. 100 nm fluorescent beads were used asfiducial markers to correct differences in alignment and chromatic

aberrations of detection paths (A,B). Fields of 150–500 beads were

imaged simultaneously in Dronpa channel (green) and Alexa Fluor

647 channel (red) and representative part of 262 mm is shown.Center positions of beads were determined from Gaussian fitting

and corresponding pairs of positions assigned. Localization

precision for beads was found 7.6 and 6.1 nm for Dronpa (green)

and Alexa Fluor 647 (red) channel, respectively (C), Pairs of center

positions were used to calculate a local weighted mean

transformation needed to correct images (D,E). Colocalization

precision of 17620 nm (mean 6 StD, n = 1857) as measured fromdistances between bead positions after application of transforma-

tion (F).

(TIF)

Figure S4 Incorporation of PAFP into HIV-1 virions andsize histograms. A) Immunofluorescence analysis (anti-CA, red)of HIV-1 virions containing indicated PAFP (green), scale bar

1 mm. B) Size distribution of Dronpa-Vpr, Gag-Dronpa and Gag-mEosFP in HIV-1 virions from super-resolution imaging and

cluster analysis. C) Size distribution of integrase (IN), capsid (CA),

matrix (MA) and Envelope (ENV) in HIV-1 virions from super-

resolution imaging and cluster analysis.

(TIF)

Figure S5 Calibration of cluster size analysis. (A)Examples of simulated fields of 25 circular cluster of different

sizes containing 100 random localizations, scale bar 1 mm, (B)Ripley’s L analysis of simulated clusters: Note decrease of peaks

and shift of maxima towards larger r at larger cluster sizes, (C)

Sizes of simulated clusters were estimated using Cluster analysis or

Ripley’s L maxima, error bars represent StD from.6 fields. (D)Sizes of simulated clusters with varied number of localizations per

cluster were estimated using cluster analysis. Error bars represent

standard deviations from measurements on six fields. Note that

there is no effect of number of localizations on mean cluster size

(TIF)

Figure S6 Characterisation of tetherin mutants. (A) 293Tcells were transfected with HIV-1 Dvpu and either tetherin-HA,tetherin-HA delTM or tetherin-HA delGPI without or with Vpu

as indicated and infectious output was determined on HeLa

indicator cells. Error bars represent range of duplicate titrations.

(B) Western blot analysis of cell lysates from A) was performed for

pr55Gag, tetherin, Vpu, GFP as transfection control and PCNA as

loading control. Note that data shown in panel A and B were

obtained under identical conditions as Figure 2 B,C. (C) 293T cells



were transfected with HIV-1 Dvpu and either HA-tetherin ormEosFP-tetherin. Virions that were constitutively released (const.)

or released following incubation of cells with subtilisin A (SubtA)

were pelleted through sucrose. Virions and corresponding cell

lysates were analyzed by quantitative Western blotting with anti-

HA and anti-p24CA antibodies. Numbers below each lane

indicate integrated densities of p24CA signal in arbitrary units.

Sizes of molecular weight markers in B and C are shown in

kilodaltons. (D) Histogram of number of mEosFP-tetherin clusters

per HIV-1 budding site. Error bars represent standard deviations

from quantification in 5 individual super-resolution images with a

total of 550 tetherin-positive HIV-1 budding sites analyzed.

(TIF)

Figure S7 Single molecule photo-physical characteriza-tion of mEosFP. (A) SDS-PAGE and Coomassie staining of 1 or4 mg of purified 6xHis-tagged mEosFP. Sizes of molecular weightmarkers are shown in kilodaltons. The predicted size of 6xHis

mEosFP is 31 kDa and lower molecular weight bands represent

mEosFP cleavage products associated with premature photoacti-

vation. (B) Representative single molecule traces of mEosFP

molecules in 1% PVA under 0.4 kW/cm2 561 nm excitation and

pulsed (red) or continuous (black) photoactivation (PA) by 405 nm

light (0.2–0.5 kW/cm2). Traces comprise 15 000 frames acquired

with 50 ms/frame. (C) Determination of dark times (td) of the

photoactivated red form of mEosFP under continuous photoac-

tivation. Number of reactivation events per single molecule trace

of (total of 15000 frames with 50 ms/frame) were determined from

25–50 traces for different td and 405 nm intensities. (D) Histogram

of number of reactivation events per single molecule trace

acquired under pulsed photoactivation (PA) or continuous PA

using 405 nm light (0.5–1.8 W/cm2) and analyzed using td = or

5 s. (E) Effect of excitation light on quantification of single mEosFP

molecules in 1% PVA. The red form of mEosFP was detected

under continuous 561 nm excitation (0.4 kW/cm2). Continuous

photoactivation by 405 nm light (1.8 W/cm2) was switched on

after 20–500 s corresponding to usual acquisition times used.

Representative regions of super-resolution images are shown.

Scale bar 1 mm. (F) Cluster analysis revealed similar number ofdetected mEosFP molecules following different exposure intervals

with 561 nm excitation light as in (E). Scale bar 1 mm(TIF)

Acknowledgments

We thank F. Leuba and A. Deres for technical assistance, F. D. Bushman,

J. Wiedenmann, V. Verkhusha, A. Miyawaki and P. Bieniasz for the kind

gift of reagents and Louise Kemp and Silvia Anghel for critical reading of

the manuscript.

Author Contributions

Conceived and designed the experiments: ML JH VP. Performed the

experiments: ML SR BM FB HU. Analyzed the data: ML SR BM FB HU.

Contributed reagents/materials/analysis tools: SR BM HU. Wrote the

paper: ML VP. Build set-up: SR HU ML.

References

1. Neil SJ, Eastman SW, Jouvenet N, Bieniasz PD (2006) HIV-1 Vpu promotes

release and prevents endocytosis of nascent retrovirus particles from the plasma

membrane. PLoS Pathog 2: e39.

2. Van Damme N, Goff D, Katsura C, Jorgenson RL, Mitchell R, et al. (2008) The

interferon-induced protein BST-2 restricts HIV-1 release and is downregulated

from the cell surface by the viral Vpu protein. Cell Host Microbe 3: 245–252.

3. Jouvenet N, Neil SJ, Zhadina M, Zang T, Kratovac Z, et al. (2009) Broad-

spectrum inhibition of retroviral and filoviral particle release by tetherin. J Virol

83: 1837–1844.

4. Sakuma T, Noda T, Urata S, Kawaoka Y, Yasuda J (2009) Inhibition of Lassa

and Marburg virus production by tetherin. J Virol 83: 2382–2385.

5. Mansouri M, Viswanathan K, Douglas JL, Hines J, Gustin J, et al. (2009)

Molecular mechanism of BST2/tetherin downregulation by K5/MIR2 of

Kaposi’s sarcoma-associated herpesvirus. J Virol 83: 9672–9681.

6. Neil SJ, Zang T, Bieniasz PD (2008) Tetherin inhibits retrovirus release and is

antagonized by HIV-1 Vpu. Nature 451: 425–430.

7. Jia B, Serra-Moreno R, Neidermyer W, Rahmberg A, Mackey J, et al. (2009)

Species-specific activity of SIV Nef and HIV-1 Vpu in overcoming restriction by

tetherin/BST2. PLoS Pathog 5: e1000429.

8. Sauter D, Schindler M, Specht A, Landford WN, Munch J, et al. (2009)

Tetherin-driven adaptation of Vpu and Nef function and the evolution of

pandemic and nonpandemic HIV-1 strains. Cell Host Microbe 6: 409–421.

9. Gupta RK, Mlcochova P, Pelchen-Matthews A, Petit SJ, Mattiuzzo G, et al.

(2009) Simian immunodeficiency virus envelope glycoprotein counteracts

tetherin/BST-2/CD317 by intracellular sequestration. Proc Natl Acad

Sci U S A 106: 20889–20894.

10. Le Tortorec A, Neil SJ (2009) Antagonism to and intracellular sequestration of

human tetherin by the human immunodeficiency virus type 2 envelope

glycoprotein. J Virol 83: 11966–11978.

11. Kupzig S, Korolchuk V, Rollason R, Sugden A, Wilde A, et al. (2003) Bst-2/

HM1.24 is a raft-associated apical membrane protein with an unusual topology.

Traffic 4: 694–709.

12. Hinz A, Miguet N, Natrajan G, Usami Y, Yamanaka H, et al. (2010) Structural

basis of HIV-1 tethering to membranes by the BST-2/tetherin ectodomain. Cell

Host Microbe 7: 314–323.

13. Perez-Caballero D, Zang T, Ebrahimi A, McNatt MW, Gregory DA, et al.

(2009) Tetherin inhibits HIV-1 release by directly tethering virions to cells. Cell

139: 499–511.

14. Hammonds J, Wang JJ, Yi H, Spearman P (2010) Immunoelectron microscopic

evidence for Tetherin/BST2 as the physical bridge between HIV-1 virions and

the plasma membrane. PLoS Pathog 6: e1000749.

15. Fitzpatrick K, Skasko M, Deerinck TJ, Crum J, Ellisman MH, et al. (2010)

Direct restriction of virus release and incorporation of the interferon-induced

protein BST-2 into HIV-1 particles. PLoS Pathog 6: e1000701.

16. McNatt MW, Zang T, Hatziioannou T, Bartlett M, Fofana IB, et al. (2009)

Species-specific activity of HIV-1 Vpu and positive selection of tetherin

transmembrane domain variants. PLoS Pathog 5: e1000300.

17. Mitchell RS, Katsura C, Skasko MA, Fitzpatrick K, Lau D, et al. (2009) Vpu

antagonizes BST-2-mediated restriction of HIV-1 release via beta-TrCP and

endo-lysosomal trafficking. PLoS Pathog 5: e1000450.

18. Goffinet C, Allespach I, Homann S, Tervo HM, Habermann A, et al. (2009)

HIV-1 antagonism of CD317 is species specific and involves Vpu-mediated

proteasomal degradation of the restriction factor. Cell Host Microbe 5: 285–297.

19. Mangeat B, Gers-Huber G, Lehmann M, Zufferey M, Luban J, et al. (2009)

HIV-1 Vpu neutralizes the antiviral factor Tetherin/BST-2 by binding it and

directing its beta-TrCP2-dependent degradation. PLoS Pathog 5: e1000574.

20. Bieniasz PD (2009) The cell biology of HIV-1 virion genesis. Cell Host Microbe

5: 550–558.

21. Betzig E, Patterson GH, Sougrat R, Lindwasser OW, Olenych S, et al. (2006)

Imaging intracellular fluorescent proteins at nanometer resolution. Science 313:

1642–1645.

22. Hess ST, Girirajan TP, Mason MD (2006) Ultra-high resolution imaging by

fluorescence photoactivation localization microscopy. Biophys J 91: 4258–4272.

23. Rust MJ, Bates M, Zhuang X (2006) Sub-diffraction-limit imaging by stochastic

optical reconstruction microscopy (STORM). Nat Methods 3: 793–795.

24. Heilemann M, van de Linde S, Schuttpelz M, Kasper R, Seefeldt B, et al. (2008)

Subdiffraction-resolution fluorescence imaging with conventional fluorescent

probes. Angew Chem Int Ed Engl 47: 6172–6176.

25. Bates M, Huang B, Dempsey GT, Zhuang X (2007) Multicolor super-resolution

imaging with photo-switchable fluorescent probes. Science 317: 1749–1753.

26. Shroff H, Galbraith CG, Galbraith JA, White H, Gillette J, et al. (2007) Dual-

color superresolution imaging of genetically expressed probes within individual

adhesion complexes. Proc Natl Acad Sci U S A 104: 20308–20313.

27. Huang B, Babcock H, Zhuang X (2010) Breaking the diffraction barrier: super-

resolution imaging of cells. Cell 143: 1047–1058.

28. Manley S, Gillette JM, Patterson GH, Shroff H, Hess HF, et al. (2008) High-

density mapping of single-molecule trajectories with photoactivated localization

microscopy. Nat Methods 5: 155–157.

29. Larson DR, Johnson MC, Webb WW, Vogt VM (2005) Visualization of

retrovirus budding with correlated light and electron microscopy. Proc Natl

Acad Sci U S A 102: 15453–15458.

30. Jouvenet N, Bieniasz PD, Simon SM (2008) Imaging the biogenesis of individual

HIV-1 virions in live cells. Nature 454: 236–240.

31. Flors C, Hotta J, Uji-i H, Dedecker P, Ando R, et al. (2007) A stroboscopic

approach for fast photoactivation-localization microscopy with Dronpa mutants.

J Am Chem Soc 129: 13970–13977.

32. Churchman LS, Okten Z, Rock RS, Dawson JF, Spudich JA (2005) Single

molecule high-resolution colocalization of Cy3 and Cy5 attached to macromol-



ecules measures intramolecular distances through time. Proc Natl Acad Sci U S A

102: 1419–1423.33. Briggs JA, Wilk T, Welker R, Krausslich HG, Fuller SD (2003) Structural

organization of authentic, mature HIV-1 virions and cores. EMBO J 22:

1707–1715.34. Carlson LA, de Marco A, Oberwinkler H, Habermann A, Briggs JA, et al. (2010)

Cryo electron tomography of native HIV-1 budding sites. PLoS Pathog 6:e1001173.

35. Zhu P, Liu J, Bess J, Jr., Chertova E, Lifson JD, et al. (2006) Distribution and

three-dimensional structure of AIDS virus envelope spikes. Nature 441:847–852.

36. Hess ST, Gould TJ, Gudheti MV, Maas SA, Mills KD, et al. (2007) Dynamicclustered distribution of hemagglutinin resolved at 40 nm in living cell

membranes discriminates between raft theories. Proc Natl Acad Sci U S A104: 17370–17375.

37. Lillemeier BF, Mortelmaier MA, Forstner MB, Huppa JB, Groves JT, et al.

(2009) TCR and Lat are expressed on separate protein islands on T cellmembranes and concatenate during activation. Nat Immunol 11: 90–96.

38. Greenfield D, McEvoy AL, Shroff H, Crooks GE, Wingreen NS, et al. (2009)Self-organization of the Escherichia coli chemotaxis network imaged with super-

resolution light microscopy. PLoS Biol 7: e1000137.

39. Annibale P, Vanni S, Scarselli M, Rothlisberger U, Radenovic A (2011)Quantitative photo activated localization microscopy: unraveling the effects of

photoblinking. PLoS One 6: e22678.40. Wiedenmann J, Ivanchenko S, Oswald F, Schmitt F, Rocker C, et al. (2004)

EosFP, a fluorescent marker protein with UV-inducible green-to-red fluores-cence conversion. Proc Natl Acad Sci U S A 101: 15905–15910.

41. Annibale P, Vanni S, Scarselli M, Rothlisberger U, Radenovic A (2011)

Identification of clustering artifacts in photoactivated localization microscopy.Nat Methods 8: 527–528.

42. Jones SA, Shim SH, He J, Zhuang X (2011) Fast, three-dimensional super-resolution imaging of live cells. Nat Methods 8: 499–508.

43. Heilemann M, van de Linde S, Mukherjee A, Sauer M (2009) Super-resolution

imaging with small organic fluorophores. Angew Chem Int Ed Engl 48:6903–6908.

44. Sougrat R, Bartesaghi A, Lifson JD, Bennett AE, Bess JW, et al. (2007) Electrontomography of the contact between T cells and SIV/HIV-1: implications for

viral entry. PLoS Pathog 3: e63.45. Ivanchenko S, Godinez WJ, Lampe M, Krausslich HG, Eils R, et al. (2009)

Dynamics of HIV-1 assembly and release. PLoS Pathog 5: e1000652.

46. Leung K, Kim JO, Ganesh L, Kabat J, Schwartz O, et al. (2008) HIV-1assembly: viral glycoproteins segregate quantally to lipid rafts that associate

individually with HIV-1 capsids and virions. Cell Host Microbe 3: 285–292.47. Habermann A, Krijnse-Locker J, Oberwinkler H, Eckhardt M, Homann S, et al.

(2010) CD317/tetherin is enriched in the HIV-1 envelope and downregulated

from the plasma membrane upon virus infection. J Virol 84: 4646–4658.48. Miyagi E, Andrew AJ, Kao S, Strebel K (2009) Vpu enhances HIV-1 virus

release in the absence of Bst-2 cell surface down-modulation and intracellulardepletion. Proc Natl Acad Sci U S A 106: 2868–2873.

49. Rollason R, Korolchuk V, Hamilton C, Jepson M, Banting G (2009) A CD317/tetherin-RICH2 complex plays a critical role in the organization of the subapical

actin cytoskeleton in polarized epithelial cells. J Cell Biol 184: 721–736.

50. Andrew AJ, Kao S, Strebel K (2011) The C-terminal hydrophobic region in

human BST-2/tetherin functions as a second transmembrane motif. J Biol

Chem 286: 39967–39981.

51. Pais-Correia AM, Sachse M, Guadagnini S, Robbiati V, Lasserre R, et al. (2009)

Biofilm-like extracellular viral assemblies mediate HTLV-1 cell-to-cell transmis-

sion at virological synapses. Nat Med 16: 83–89.

52. Masuyama N, Kuronita T, Tanaka R, Muto T, Hirota Y, et al. (2009) HM1.24

is internalized from lipid rafts by clathrin-mediated endocytosis through

interaction with alpha-adaptin. J Biol Chem 284: 15927–15941.

53. Nguyen DH, Hildreth JE (2000) Evidence for budding of human immunode-

ficiency virus type 1 selectively from glycolipid-enriched membrane lipid rafts.

J Virol 74: 3264–3272.

54. Ono A, Freed EO (2001) Plasma membrane rafts play a critical role in HIV-1

assembly and release. Proc Natl Acad Sci U S A 98: 13925–13930.

55. Miyagi E, Andrew A, Kao S, Yoshida T, Strebel K (2011) Antibody-mediated

enhancement of HIV-1 and HIV-2 production from BST-2/tetherin+ cells.J Virol 85: 11981–11994.

56. Lingwood D, Simons K (2010) Lipid rafts as a membrane-organizing principle.

Science 327: 46–50.

57. van Zanten TS, Gomez J, Manzo C, Cambi A, Buceta J, et al. (2010) Direct

mapping of nanoscale compositional connectivity on intact cell membranes.

Proc Natl Acad Sci U S A 107: 15437–15442.

58. Hogue IB, Grover JR, Soheilian F, Nagashima K, Ono A (2011) Gag Induces

the Coalescence of Clustered Lipid Rafts and Tetraspanin-Enriched Micro-

domains at HIV-1 Assembly Sites on the Plasma Membrane. J Virol 85:

9749–9766.

59. Krementsov DN, Rassam P, Margeat E, Roy NH, Schneider-Schaulies J, et al.

(2010) HIV-1 assembly differentially alters dynamics and partitioning of

tetraspanins and raft components. Traffic 11: 1401–1414.

60. Naldini L, Blomer U, Gallay P, Ory D, Mulligan R, et al. (1996) In vivo gene

delivery and stable transduction of nondividing cells by a lentiviral vector.

Science 272: 263–267.

61. Schneider R, Campbell M, Nasioulas G, Felber BK, Pavlakis GN (1997)

Inactivation of the human immunodeficiency virus type 1 inhibitory elements

allows Rev-independent expression of Gag and Gag/protease and particle

formation. J Virol 71: 4892–4903.

62. Ando R, Mizuno H, Miyawaki A (2004) Regulated fast nucleocytoplasmic

shuttling observed by reversible protein highlighting. Science 306: 1370–1373.

63. Habuchi S, Tsutsui H, Kochaniak AB, Miyawaki A, van Oijen AM (2008)

mKikGR, a monomeric photoswitchable fluorescent protein. PLoS One 3:

e3944.

64. Chudakov DM, Lukyanov S, Lukyanov KA (2007) Tracking intracellular

protein movements using photoswitchable fluorescent proteins PS-CFP2 and

Dendra2. Nat Protoc 2: 2024–2032.

65. Subach FV, Malashkevich VN, Zencheck WD, Xiao H, Filonov GS, et al. (2009)

Photoactivation mechanism of PAmCherry based on crystal structures of the

protein in the dark and fluorescent states. Proc Natl Acad Sci U S A 106:

21097–21102.

66. Wendy L, Martinez ARM (2008) Computational Statistics Handbook with

MATLAB. Chapman & Hall/CRC.



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